Essential role of MD-2 in TLR4-dependent signaling during Helicobacter pylori- associated gastritis

toll样受体4（Toll-like receptor 4, TLR4）是一种模式识别受体，是脂多糖（LPS）的主要受体。MD-2物理上与细胞表面的TLR4结合，并赋予LPS反应性。幽门螺杆菌LPS是诱发胃炎的主要毒力因子之一。我们在这里证明了MD-2在幽门螺杆菌相关胃炎中tlr4依赖性信号传导中的作用。本研究使用了来自幽门螺杆菌感染患者和未感染患者以及4种胃癌细胞系的胃活检样本。RT-PCR检测活检标本和细胞系中TLR-4和MD-2的表达。免疫组织化学检测组织切片中TLR-4的定位。为了进行体外功能测试，我们建立了稳定的表达TLR4和MD-2的AGS细胞转染物。流式细胞术检测TLR4的细胞分布。采用报告基因法检测NF-κB活化、IL-8和MD-2启动子活化情况。幽门螺杆菌感染可上调胃黏膜TLR4和MD-2的表达。TLR4染色主要见于上皮细胞，位于细胞质和根尖表面。在幽门螺杆菌LPS刺激下，MD-2转染AGS细胞可显著提高细胞表面TLR4的表达，增强NF-κB和IL-8启动子的活化。活的幽门螺杆菌也刺激了MD-2的转录激活。本研究发现，在幽门螺杆菌感染期间，胃上皮细胞中MD-2的表达升高，表明TLR4/MD-2系统是一个有效的受体复合物，参与了胃对幽门螺杆菌LPS的反应。

Toll-like receptor 4(TLR4), a member of pattern recognition receptors, is the main receptor of lipopolysaccharide(LPS). MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. H.pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated here the role of MD-2 in TLR4-dependent signaling in H.pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H.pylori infection and 4 gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-κB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H.pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-κB and IL-8 promoter upon stimulation with H.pylori LPS. Live H.pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H.pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H.pylori LPS in the stomach.